KRT13 Monoclonal Antibody

What is KRT13 and what is its biological significance?

KRT13 (Keratin 13) is a type I intermediate filament protein specifically expressed in stratified squamous epithelia, including the oral mucosa, esophagus, and female genital tract. It forms filaments that provide mechanical stability to epithelial cells, maintaining structural integrity of tissues and creating barriers against physical and chemical stress. Beyond its structural role, KRT13 plays important functions in cell signaling and regulation of gene expression . Recent research has revealed its significant role in cancer progression and metastasis, particularly in breast cancer where it promotes tumor growth and metastatic behavior through interaction with plakoglobin (PG) and subsequent modulation of c-Myc signaling .

How are KRT13 monoclonal antibodies produced and purified?

KRT13 monoclonal antibodies are produced using hybridoma technology through the following process:

• Immunization: Mice are immunized with synthesized peptides derived from human KRT13

• Cell fusion: B cells from immunized mice spleens are isolated and fused with myeloma cells to create hybridomas

• Screening: Hybridomas are screened to select those producing antibodies specific to KRT13

• Cultivation: Selected hybridomas are cultured in mouse abdominal cavity

• Purification: KRT13 monoclonal antibodies are purified from mouse ascites by affinity chromatography using specific immunogens

The resulting purified antibodies are highly specific, reacting exclusively with human KRT13 protein in applications such as ELISA and immunohistochemistry (IHC) .

What applications are KRT13 monoclonal antibodies commonly used for?

KRT13 monoclonal antibodies can be utilized in multiple research applications:

The optimal working dilution should be determined experimentally for each specific application and tissue type .

How should KRT13 monoclonal antibodies be stored to maintain activity?

For optimal preservation of antibody activity:

• Store at -20°C to -80°C

• Aliquot the antibody solution to avoid repeated freezing and thawing cycles

• Most commercial KRT13 antibodies are supplied in buffer systems like 10 mM PBS

• Avoid prolonged exposure to room temperature or direct light

• Follow manufacturer's specific storage recommendations for each antibody preparation

Proper storage is critical for maintaining antibody specificity and preventing degradation that could compromise experimental results.

How can KRT13 monoclonal antibodies be used to study cancer progression and metastasis?

KRT13 has emerged as an important marker and functional driver of cancer progression, particularly in breast cancer. Researchers can utilize KRT13 monoclonal antibodies to:

Methodologically, immunohistochemical analysis of KRT13 should combine assessment of both percentage of positive cells and staining intensity for comprehensive scoring .

What are the common technical challenges when using KRT13 antibodies and how can they be overcome?

Several technical challenges may arise when working with KRT13 monoclonal antibodies:

• Cross-reactivity with other keratins: Some antibodies may cross-react with similar keratins. Solution: Use well-characterized monoclonal antibodies with confirmed specificity, such as clone DE-K13

• Variable expression in xenograft models: KRT13 overexpression may appear uneven in xenograft tumors compared to in vitro cultures. Solution: Incorporate multiple detection methods and sampling locations

• Edge-positive phenomenon: KRT13 often shows stronger staining at extending edges of cell colonies. Solution: Carefully document this pattern and incorporate it into analysis rather than considering it an artifact

• Antibody specificity in different applications: An antibody working well for IHC may not perform optimally in Western blotting. Solution: Validate each antibody for specific applications and optimize protocols accordingly

• Fixation sensitivity: Some epitopes may be masked by certain fixation methods. Solution: Test multiple fixation protocols or use epitope retrieval methods if necessary

How can KRT13 expression be effectively quantified in tissue samples?

Accurate quantification of KRT13 expression is critical for research validity:

• Immunohistochemistry scoring systems:

  • Combine percentage of positive cells and staining intensity

  • Use digital image analysis software for objective quantification

  • Consider spatial distribution, especially focusing on the invasive front in tumors

• Protein quantification:

  • Use Western blotting with proper controls

  • Perform morphometric analysis with software like Image Studio

  • Include multiple loading controls for normalization

• RNA expression analysis:

  • Extract RNA using RNeasy Mini Kit or similar high-quality extraction methods

  • Perform RNA-seq or qPCR with appropriate reference genes

  • Consider comparing expression to cancer genome databases like TCGA

• Single-cell analysis:

  • Use flow cytometry for quantifying KRT13 at the single-cell level

  • Consider single-cell RNA-seq for heterogeneity assessment

What experimental models are most appropriate for studying KRT13 function?

Selection of appropriate experimental models is crucial for meaningful KRT13 research:

• Cell line models:

  • Use paired low/high KRT13-expressing cell lines (e.g., MCF7 with low endogenous KRT13 and HCC1954 with high endogenous KRT13)

  • Generate stable KRT13 overexpression lines using lentiviral systems (e.g., pLVX-AcGFP1-N1 vector)

  • Create KRT13 knockdown lines using shRNA lentiviral particles

• Animal models:

  • Orthotopic xenograft models (e.g., mammary fat pad injection for breast cancer studies)

  • Intracardiac injection models for studying metastasis

  • Patient-derived xenografts for higher clinical relevance

• 3D culture systems:

  • Organoid cultures to better recapitulate epithelial organization

  • 3D matrix invasion assays to study KRT13's role in invasion/migration

• Validation in human specimens:

  • Correlate findings with human tissue microarrays

  • Consider multiplexed quantum dot labeling for co-expression studies

How does KRT13 interact with plakoglobin and what are the downstream effects?

KRT13 has been shown to interact with plakoglobin (PG, also known as γ-catenin) in a complex molecular mechanism:

• Physical interaction: KRT13 directly interacts with plakoglobin to form complexes with desmoplakin (DSP)

• Effects on plakoglobin function:

  • Interferes with PG expression levels

  • Inhibits nuclear translocation of PG

  • Abrogates PG-mediated suppression of c-Myc expression

• Downstream signaling consequences:

  • Activation of the KRT13/PG/c-Myc signaling pathway

  • Increased epithelial-to-mesenchymal transition (EMT)

  • Enhanced stem cell-like phenotype in cancer cells

• Cellular fractionation studies: To investigate this mechanism, researchers should perform careful cell fractionation to separate cytoplasmic and nuclear extracts using nuclear and cytoplasmic extraction reagents, followed by Western blot analysis of key proteins in each fraction

What methods can be used to study the epithelial-to-mesenchymal transition (EMT) mediated by KRT13?

EMT is a critical process in cancer progression that KRT13 has been shown to promote. Researchers can investigate KRT13-mediated EMT using:

• Morphological assessment:

  • Monitor changes in cell shape and organization

  • Track formation of extending edges in colony assays

• Protein marker analysis:

  • Assess epithelial markers (E-cadherin, cytokeratins) and mesenchymal markers (N-cadherin, vimentin)

  • Use Western blotting, immunofluorescence, and flow cytometry for comprehensive analysis

• Functional assays:

  • Cell migration assays (wound healing, transwell)

  • Invasion assays using Matrigel or other ECM components

  • Single-cell tracking to measure motility parameters

• Gene expression profiling:

  • RNA-seq to identify EMT-related transcriptional changes

  • Compare to established EMT gene signatures

  • Analyze correlation with breast cancer molecular subtypes

• Stemness assessment:

  • Evaluate cancer stem cell markers (CD44, ALDH1A1, Nanog)

  • Perform sphere formation assays

  • Analyze correlation between KRT13, EMT, and stemness markers

How does KRT13 expression correlate with clinical outcomes in cancer patients?

KRT13 expression has significant clinical implications, particularly in breast cancer:

Can KRT13 serve as a therapeutic target for cancer treatment?

The involvement of KRT13 in promoting breast cancer progression and metastasis suggests its potential as a therapeutic target:

• Targeting strategies:

  • Direct inhibition of KRT13 expression (e.g., siRNA, shRNA approaches)

  • Disruption of KRT13-PG interaction

  • Modulation of downstream pathways (c-Myc inhibition)

• Combination approaches:

  • Combining KRT13-targeted therapies with conventional treatments

  • Targeting multiple nodes in the KRT13/PG/c-Myc pathway

• Biomarker for therapy selection:

  • KRT13 expression might identify patients likely to benefit from specific therapies

  • Its expression at the invasive front could guide surgical margins

• Challenges to consider:

  • KRT13's normal physiological role in epithelial integrity

  • Potential off-target effects

  • Delivery of targeting agents to the tumor site

Research suggests that targeting the KRT13-mediated pathway represents a potential novel approach for therapeutic intervention in breast cancer progression and metastasis .

What is the role of KRT13 in cancer stemness and how can it be investigated?

Recent research has uncovered KRT13's involvement in promoting cancer stemness:

• Stemness markers:

  • KRT13 overexpression correlates with increased expression of stemness markers like CD44, ALDH1A1, and Nanog

  • These markers can be detected by Western blotting, immunofluorescence, and flow cytometry

• Functional assays:

  • Sphere formation assays to assess self-renewal capacity

  • Serial dilution transplantation to evaluate tumor-initiating potential

  • Drug resistance assays, as stem-like cells often show increased resistance

• Signaling pathway analysis:

  • Focus on the KRT13/PG/c-Myc axis, as c-Myc is a known regulator of stemness

  • Investigate relationships with other stemness-related pathways (Wnt, Notch, Hedgehog)

• Single-cell approaches:

  • Single-cell RNA-seq to identify stemness signatures in KRT13-high cells

  • Analyze cellular heterogeneity within tumors and correlation with KRT13 expression

• Therapeutic implications:

  • Evaluate strategies targeting both KRT13 and stemness pathways

  • Assess impact on tumor recurrence and therapy resistance

How do post-translational modifications affect KRT13 function in normal and pathological conditions?

Post-translational modifications (PTMs) likely play a critical role in regulating KRT13 function:

• Types of PTMs to investigate:

  • Phosphorylation, which often regulates intermediate filament assembly/disassembly

  • Glycosylation, which may affect protein stability and interactions

  • Ubiquitination, which regulates protein turnover

  • Acetylation and other modifications that might affect binding properties

• Detection methods:

  • Mass spectrometry to identify specific PTM sites

  • Phospho-specific antibodies for phosphorylation studies

  • 2D gel electrophoresis to separate modified protein forms

• Functional consequences:

  • Effects on filament formation and stability

  • Altered binding to interaction partners like plakoglobin

  • Changes in subcellular localization and turnover

• Temporal and spatial regulation:

  • Different modifications in normal versus cancer tissues

  • Modifications at the invasive front versus tumor core

  • Changes during cancer progression and metastasis

Understanding PTMs could provide new insights into KRT13 regulation and identify additional therapeutic targeting opportunities.